Membrane transport systems are involved in a number of fundamental cellular processes, and yet are poorly characterized at the molecular level. The glucose transporter is the prototype facilitated diffusion transport protein. Facilitative glucose transport is a critical function carried out by virtually all mammalian cells. This process is mediated by a family of tissuespecific membrane glycoproteins containing five isoforms. These proteins are responsible for the net uptake of glucose from the blood into cells, supplying cellular glucose for energy me- tabolism and the biosynthesis of sugar-containing macromolecules. Additionally, glucose transport in certain tissues plays a critical role in organismal glucose homeostasis. It has been suggested that a defect in one or more glucose transporter genes could predispose to non-insu- lin-dependent diabetes. Despite the physiologic importance of glucose transport, little is known concerning the structure and function of these proteins. The long-term goal of the studies described in this proposal is to understand the molecular mechanism of glucose transport. As an initial step towards this goal, the following aims will be undertaken: 1) A topological model has been proposed for the glucose transporters based on their deduced primary structures and a limited amount of structural data available for the human erythrocyte protein. The membrane topology of the GLUT 1 transporter will be determined by constructing chimeric molecules containing markers within the predicted soluble domains. One marker will be the consensus sequence for N-linked glycosylation, and the other marker will be the highly antigenic poliovirus C3 epitope. An in vitro translation/translocation system and a mammalian transfection system will be used to express the chimeric molecules. 'ne disposition of the markers with respect to the membrane will be determined using immunologic and enzymatic assays. 2) Site-directed mutagenesis of a cDNA clone and functional expression of mutant transporters in Xenopus oocytes will be used to localize the cytochalasin B-binding site of the GLUT 1 protein and to define amino acid residues directly involved in transport function.